Project Summary The goal of this exploratory research proposal is to determine targeted therapeutic options for acute myeloid leukemia (AML) that are presented by the activity of APOBEC3A, an endogenous DNA mutator. APOBEC3 cytosine deaminases are best defined by their ability to mutate viral DNA, thereby restricting viral infection. Through off-target activity, APOBEC3 enzymes also have the capacity to mutate cellular DNA and have been implicated as the source of specific mutational patterns identified in cancer genome sequences. One member of the APOBEC3 family, APOBEC3A, is highly expressed in myeloid lineage cells. Approximately 15 percent of pediatric and adult AML exhibit extremely high expression levels of APOBEC3A. Recent data indicate that APOBEC3A activity on the cancer genome triggers cellular DNA damage responses, specifically ATR and Chk1 kinases involved in the replication stress response. When ATR and Chk1 are inhibited, APOBEC3A activity results in genotoxicity and cancer cell death. These in vitro data serve as a paradigm for the goals of the proposed studies: (1) to identify additional cellular pathways activated by APOBEC3A activity that pose potential therapeutic options in AML, and (2) to determine the impact of APOBEC3A activity on AML in a model organism. In the proposed research, a genome-wide loss-of-function screen will be performed to determine cellular factors that synergize with APOBEC3A activity to cause cancer cell death. The experimental approach for this screen is through utilization of a genome-wide CRISPR guide RNA library in combination with APOBEC3A activity, which will enable high-throughput viability screening of individual gene knock-outs in AML cell lines. Screen results will be validated and prioritized by targeted knock-out of identified genes, and chemical inhibition of protein products in AML cells with high A3A expression. Genetic and chemical inhibition will be performed in a panel of AML cell lines with high APOBEC3A expression, and then validated in primary AML cells. Additionally, the proposed studies will generate xenograft mouse models using AML cell lines and primary AML cells with high APOBEC3A expression. Animal models will be utilized to evaluate the in vivo impact of APOBEC3A activity in AML on disease progression and overall survival. Pre-clinical testing of chemicals identified to synergize with APOBEC3A activity will be done using xenograft mouse models. These studies have the potential to uncover synthetic lethal interactions that can be exploited for therapeutic targeting of AML in which APOBEC3A is active.